Inorganic polyphosphate essential for lytic growth of phages P1 and fd

Abstract

Transduction frequency with phage P1 had been observed to be very low in Escherichia coli K-12 mutants lacking the operon (ppk1-ppx) responsible for the synthesis of inorganic polyphosphate (poly P). We now find that these mutants, for lack of poly P, are lysogenic for P1 and when infected with phage P1 produce only Ϸ1% the number of infective centers compared with the WT host. Both phage adsorption and release were unaffected. The hostencoded P1 late-gene transcriptional activator, SspA, failed to show the transcriptional increase in the mutant, observed in the WT. UV induction of a P1-infected mutant resulted in a 200-fold increase in the production of infectious phage particles. The lysogenized P1 (P1mut) and P1 progeny from the mutant host (⌬ppk1-ppx) produced plaques of differing morphologies, whereas P1 progeny from the WT yielded only small, clear plaques. Two discernable variants, one producing small and clear plaques (P1small) and the other large plaques with turbid rims (P1large), had broader host range and produced larger burst sizes in WT compared with P1. Transmission electron microscopy showed P1mut had contractile sheath defects. Thus, the lack of poly P/PPK1 in the mutant host resulted in the formation of defective P1 particles during intracellular growth. A filamentous phage, fd, also failed to produce plaques on a mutant lawn. Although fd adsorbed to the F-pilus, its DNA failed to enter the mutant host.contractile sheath ͉ lysogeny ͉ lytic replication ͉ Stringent Starvation protein A I norganic polyphosphate (poly P), a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds, ranges in length from tens to hundreds of residues. Poly P is evolutionarily conserved and has been found in all organisms (1, 2). Poly P accumulation may be very high in many bacteria and fungi, accounting for as much as 10-20% of dry weight (3, 4). Intracellular accumulation of poly P in these organisms is an essential response to stress and for survival (4, 5). The principal poly P-synthesizing enzyme in several prokaryotic species, including Escherichia coli, is polyphosphate kinase 1 (PPK1), which catalyzes the transfer of the terminal phosphate group of ATP to a growing chain of poly P (6). A null mutant of PPK1 is defective in transcription, cell motility, quorum sensing, biofilm formation, and survival (7-9). In E. coli infected with the temperate bacteriophage , the p R promoter is negatively regulated by guanosine 3Ј,5Ј-bisdiphosphate (ppGpp), at the level of transcription (10). Phage P1 is a temperate phage like , but the P1 prophage exists as an episome (11). Although its replication in E. coli under stringent response has not been specifically examined, mutants in the stringent starvation protein A (sspA) are defective in P1 lytic replication (12). Previously, we observed a very low transduction frequency (Ͻ10 Ϫ6 ) when we attempted to transfer the ppk1-ppx mutation into E. coli and Shigella spp. (4, 5). To understand how the cell physiology of the ppk1-ppx null mutant affected P...

MglA/SspA Complex Interactions Are Modulated by Inorganic Polyphosphate

“…Recent reports by Li et al . [23] and others [7], [24] indicated the essential role of polyP in stress response and stationary phase survival. We hypothesized that the higher β-galactosidase activity observed in the AW24 strain (Δ relA Δ spoT ) was directly related to higher intracellular concentrations of polyP.…”

Group II Intron Protein Localization and Insertion Sites Are Affected by Polyphosphate

“…In addition to affecting the intracellular localization of LtrA, the binding of poly(P) may directly affect LtrA's biochemical activities in RNA splicing and intron mobility. Precedents for such effects include the findings that poly(P) activates Lon protease degradation of ribosomal proteins [ 42 ], may play a role in activating mammalian TOR kinase [ 43 ], and is required for the lytic growth of phages P1 and fd [ 44 ]. Our finding that poly(P) inhibits LtrA's RT activity ( Figure 7 B) may explain why the ppk mutant, which has decreased levels of poly(P), shows increased Ll.LtrB mobility frequencies in a chromosomal lacZ gene integration assay ( Figure 5 B).…”

Neurotrophic Viruses in the Pathogenesis of ALS

“…Polyp provides phosphate groups for ATP synthesis in all life forms, from viruses, bacteria and humans. As linear polymers, they are essential for the survival of enteropathogens, including E. Coli [56] and are used by bacterium to manufacture amyloid like biofilms [57] (Figure 2). In viral disease, PolyP provides phosphate groups for RNA polymerase enzymes to assist in the fidelity of the viral genome by providing phosphate groups to their nucleotide sequences.…”